A combined drug combination for inhibiting the proliferation of NPM1 mutant leukemia cells and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a combination drug combination and application for inhibiting the proliferation of NPM1 mutant leukemia cells. The combination drug combination includes RAS selective lethal molecule 3 and sulfonamide antibiotics; and anti-tumor small molecule drugs. The sulfonamide antibiotics include sulfasalazine; the anti-tumor small molecule drugs are selected from one or more of cytarabine, daunorubicin and venetoclax. The present invention also provides the application of the pharmaceutical composition in the combination drug combination in the preparation of an NPM1 mutant leukemia ferroptosis effect evaluation reagent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a combined drug combination for inhibiting the proliferation of NPM1 mutant leukemia cells and its application. Background Art

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy originating from hematopoietic stem cells and is the most common type of acute leukemia in adults. Among them, AML with nucleophosmin (NPM1) gene mutations has been established as an independent subtype. NPM1 mutations are one of the most common gene mutations in AML, with about 35% of newly diagnosed AMLs having this gene mutation, and the proportion of NPM1 mutations in AMLs with normal karyotypes can account for 40-50%. Since NPM1 mutations often occur in AMLs with normal karyotypes, it is difficult to treat them with drugs currently used for abnormal karyotype leukemias (such as imatinib for the BCR-ABL fusion gene in chronic myeloid leukemia).

[0003] Based on the structure and function of NPM1 mutant protein, many studies have proposed potential targeted therapeutic strategies for NPM1 mutant AML. However, the efficacy and safety of targeted drugs are not very high, and there are few clinical trials of targeted therapeutic strategies. At present, the treatment methods for this subtype are still mainly chemotherapy and hematopoietic stem cell transplantation. Due to poor physical condition and poor tolerance, the treatment effect of chemotherapy is poor for elderly patients, and some young patients will also experience relapse and refractory after receiving chemotherapy. In addition, hematopoietic stem cell transplantation also faces risks such as matching difficulties and postoperative graft-versus-host disease. Some studies even point out that AML patients with NPM1 mutations are not suitable for hematopoietic stem cell transplantation. Furthermore, the emerging immunotherapy has many limitations in its application. On the one hand, the lack of tumor-specific targets on the surface of AML cells limits the application of CAR-T therapy. On the other hand, AML cells can induce immunosuppression in the tumor microenvironment through various pathways, thereby affecting the efficacy of immunotherapy. In summary, there are still many difficulties and challenges in the treatment of AML.

[0004] Developing precision treatment for the corresponding targets for the disease is more in line with the development trend of modern medicine, while the existing targeted treatment schemes still have many limitations. For example, the invention patent with publication number CN114317755A involves "the application of m6A demethylase FTO in inhibiting the proliferation of NPM1 mutant leukemia cells". The invention reveals that m6A demethylase FTO is expected to become a new molecular target for the treatment of NPM1 mutant leukemia, and verifies the ability of meclofenac sodium to inhibit the proliferation of NPM1 mutant leukemia cells and promote apoptosis of NPM1 mutant leukemia cells. However, meclofenac sodium is not efficient in inducing apoptosis, and only when the drug concentration is high will it cause more cell death, which may cause NPM1 mutant leukemia cells to develop drug resistance, making it more difficult to treat. For another example, Venetoclax (VEN), which targets the anti-apoptotic protein B cell lymphoma 2 (B cell lymphoma, BCL-2), has been shown to have a good effect on some patients with AML, but some studies have shown that the therapeutic effect of VEN alone is limited.

[0005] In summary, there is an urgent need to find new treatments and strategies to address the treatment dilemma of NPM1-mutated AML. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a combination drug combination for inhibiting the proliferation of NPM1 mutant leukemia cells and the application of the combination drug combination, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The specific technical scheme is as follows.

[0007] In one aspect, the present invention provides a novel drug combination.

[0008] The present invention provides a combination drug combination for inhibiting the proliferation of NPM1 mutant leukemia cells, wherein the combination drug combination comprises RAS selective lethal 3 (RSL3) and sulfonamide antibiotics; the sulfonamide antibiotics comprise sulfasalazine (SSZ).

[0009] Furthermore, the combination drug of the present invention also includes an anti-tumor small molecule chemical drug; the anti-tumor small molecule chemical drug is selected from at least one of cytarabine (Ara-C), daunorubicin (DNR) and venetoclax (VEN).

[0010] Furthermore, the sulfonamide antibiotic is also selected from at least one of sulfadiazine, sulfamethoxazole and sulfamethoxazole.

[0011] Furthermore, the NPM1 mutant leukemia cells are cells that highly express glutathione peroxidase 4 (GPX4).

[0012] In some preferred embodiments, the cell with high GPX4 expression is OCI-AML3.

[0013] Furthermore, the effective starting concentration of the RAS selective lethal molecule 3 in the combined drug combination is not less than 0.1 μM; the effective starting concentration of the sulfonamide antibiotic is not less than 100 μM.

[0014] On the other hand, the present invention provides an application of a pharmaceutical composition in the preparation of a reagent for evaluating the ferroptosis effect in NPM1 mutant leukemia, wherein the pharmaceutical composition comprises RAS selective lethal molecule 3 and a sulfonamide antibiotic; the sulfonamide antibiotic is selected from at least one of sulfasalazine, sulfadiazine, sulfamethoxazole and sulfamethoxazole; the reagent for evaluating the ferroptosis effect in NPM1 mutant leukemia comprises a ferroptosis inhibitor and a GPX4 expression level detection reagent.

[0015] Furthermore, the NPM1 mutant leukemia ferroptosis effect evaluation reagent also includes experimental cells, which are NPM1 mutant leukemia cells with high expression of GPX4; or, the pharmaceutical composition configured in a unit dose in the NPM1 mutant leukemia ferroptosis effect evaluation reagent.

[0016] In some preferred embodiments, the cell with high GPX4 expression is OCI-AML3.

[0017] Furthermore, the ferroptosis inhibitor is used to evaluate the level of ferroptosis induced by the sulfonamide antibiotics.

[0018] In some preferred embodiments, the ferroptosis inhibitor is selected from at least one of ferrostatin-1, lipoxstatin-1, troglitazone and zileuton.

[0019] Specifically, the sulfonamide antibiotics can induce ferroptosis in the experimental cells, and after adding the ferroptosis inhibitor, the ferroptosis process of the experimental cells can be reversed to varying degrees to reduce the level of ferroptosis induced by the sulfonamide antibiotics, thereby evaluating the efficacy of the selected sulfonamide antibiotics in inducing ferroptosis in the experimental cells.

[0020] Furthermore, the GPX4 expression level detection reagent is used to evaluate the expression level of GPX4 in NPM1 mutant leukemia cells inhibited by RAS selective lethal molecule 3.

[0021] Specifically, the GPX4 expression level detection reagent may include a real-time fluorescence quantitative PCR reagent and / or a protein immunoblotting test reagent for quantitatively detecting the gene expression level and / or protein expression level of GPX4.

[0022] Furthermore, the effective starting concentration of the RAS selective lethal molecule 3 in the pharmaceutical composition is not less than 0.1 μM; the effective starting concentration of the sulfonamide antibiotic is not less than 100 μM.

[0023] On the other hand, the present invention can also provide a more preferred combination of drugs, as follows.

[0024] The combined drug therapy includes a three-drug combination of RAS selective lethal molecule 3 and sulfonamide antibiotics combined with anti-tumor small molecule chemical drugs.

[0025] Furthermore, the sulfonamide antibiotic is selected from at least one of sulfasalazine, sulfadiazine, sulfamethoxazole and sulfamethoxazole.

[0026] As a preferred embodiment, the sulfonamide antibiotic may be sulfasalazine.

[0027] Furthermore, the anti-tumor small molecule drug is selected from at least one of cytarabine, daunorubicin and venetoclax.

[0028] Furthermore, the effective starting concentration of the RAS selective lethal molecule 3 is not less than 0.1 μM; the effective starting concentration of the sulfonamide antibiotic is not less than 100 μM.

[0029] Beneficial effects:

[0030] (1) The present invention provides a novel combination of drugs, which achieves the effect of inhibiting the proliferation of NPM1 mutant leukemia cells by combining low-dose sulfonamide antibiotics sulfasalazine and RAS selective lethal molecule 3. Specifically, the present invention provides a combination of drugs for inhibiting the proliferation of NPM1 mutant leukemia cells and the use of the drug composition in the preparation of an NPM1 mutant leukemia ferroptosis effect evaluation reagent. The present invention first proves that NPM1 mutant leukemia cells have high expression of GPX4, and proves that its expression level is related to NPM1 mutation, and further proves that RAS selective lethal molecule 3 can inhibit the proliferation of NPM1 mutant leukemia cells by inhibiting the expression level of GPX4 in NPM1 mutant leukemia cells. GPX4 is a key molecule that resists the ferroptosis process, and its downregulation can promote the occurrence of ferroptosis. Secondly, the present invention also proves that the high expression of GPX4 in NPM1 mutant leukemia cells makes them more sensitive to sulfasalazine, and can cause more NPM1 mutant leukemia cells to die at a lower concentration. And it is further confirmed that this death mode is a new type of ferroptosis. Therefore, RAS selective lethal molecule 3 and sulfasalazine can reduce the ferroptosis resistance level of NPM1 mutant leukemia cells through low-dose combination, thereby achieving the effect of increasing the level of sulfasalazine-induced ferroptosis.

[0031] (2) As a more preferred drug combination, RAS selective lethal molecule 3 and sulfasalazine combined with anti-tumor small molecule drugs have a better inhibitory effect on the proliferation of NPM1 mutant leukemia cells. Specifically, the experiment of the present invention confirmed that RAS selective lethal molecule 3 and sulfasalazine have a synergistic effect. RAS selective lethal molecule 3 further enhances the ferroptosis induced by low-dose sulfasalazine by reducing the level of ferroptosis resistance. The two synergistically further overcome the resistance of NPM1 mutant leukemia cells to anti-tumor small molecule drugs, achieving the purpose of improving the therapeutic effect and higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0033] Figure 1 The expression level of GPX4 in different leukemia cell lines in one of the embodiments;

[0034] Figure 2 This is the result of regulation of GPX4 level by NPM1 mutant protein in one of the examples;

[0035] Figure 3 The effect of SSZ on the activity of different leukemia cells and the drug sensitivity results thereof in one of the examples;

[0036] Figure 4 The effect of Erastin on the activity of different leukemia cells and the drug sensitivity results thereof in one of the examples;

[0037] Figure 5 The effect of RSL3 on the activity of different leukemia cells and the drug sensitivity results thereof in one of the examples;

[0038] Figure 6 The results of the effects of SSZ on the activities of normal cells and different leukemia cells in one of the examples;

[0039] Figure 7 The results of Erastin's effects on the activities of normal cells and different leukemia cells in one of the examples;

[0040] Figure 8 In one of the examples, the effect of RSL3 and SSZ combined with cytarabine (Ara-C) on the activity of OCI-AML3 cells;

[0041] Fig. 9 In one of the examples, the effect of RSL3 and SSZ combined with daunorubicin (DNR) on the activity of OCI-AML3 cells;

[0042] Fig.10 In one of the examples, the effect of RSL3 and SSZ combined with venetoclax (VEN) on the activity of OCI-AML3 cells;

[0043] Fig.11 The effect of the combination of RSL3 and SSZ on the activity of OCI-AML3 cells in one of the embodiments;

[0044] Fig.12 is the synergistic effect score of the combination of RSL3 and SSZ in one of the embodiments;

[0045] Fig.13 The expression level of GPX4 in OCI-AML3 cells after RSL3 treatment in one of the embodiments;

[0046] Fig.14 The result of the correlation between the sensitivity of leukemia cells to SSZ and the expression of GPX4 in one of the examples;

[0047] Fig.15This is one of the examples in which different death inhibitors restore the suppressed proliferation activity of OCI-AML3 cells after SSZ treatment. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0049] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0050] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0051] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0053] Definition of noun:

[0054] The "anti-tumor small molecule drugs" mentioned in the present invention refer to chemically synthesized drugs, which have specific chemical structures and mechanisms of action; they are different from biological macromolecule drugs (including peptides, proteins, antibodies, polysaccharides and nucleic acids, etc.).

[0055] The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The cell culture and statistical analysis methods used in the present invention are as follows:

[0056] Experimental materials and reagents:

[0057] (1) Cells: HS-5, THP-1, NB4, and HL-60 cells were purchased from the American Type Culture Collection (ATCC). OCI-AML2 and OCI-AML3 cells were purchased from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ).

[0058] (2) Reagents:

[0059] The culture medium required for the growth of NB4, HL-60, THP-1 and OCI-AML2 cell lines is prepared with RPMI-1640 culture medium, South American fetal bovine serum and penicillin-streptomycin solution (100 U / mL) at a ratio of 100:10:1. The culture medium required for the growth of OCI-AML3 cells is prepared with RPMI-1640 culture medium, Australian fetal bovine serum and penicillin-streptomycin solution (100 U / mL) at a ratio of 100:10:1. The culture medium required for the growth of HS-5 cells is prepared with DMED culture medium, South American fetal bovine serum and penicillin-streptomycin solution (100 U / mL) at a ratio of 100:10:1.

[0060] (3) Methods:

[0061] HS-5, NB4, HL-60, THP-1, OCI-AML2 and OCI-AML3 cells were cultured in an incubator at 37°C and 5% CO2. Cells were passaged every 2-3 days on average to maintain logarithmic growth.

[0062] (4) Statistical analysis:

[0063] All data were obtained from three independent experiments, and the quantitative data were expressed as mean ± standard deviation ( ±SD). GraphPad Prism (Version 8.0.1) software was used for statistical analysis. Unpaired Student's t-test was used to compare the means of two samples, and one-way analysis of variance was used to compare multiple groups of data. The test level was α=0.05, and P<0.05 indicated that the difference was statistically significant.

[0064] Example 1

[0065] This example studies the expression level of glutathione peroxidase 4 (GPX4) in leukemia cell lines.

[0066] It should be noted that there is currently no authoritative guideline that uses GPX4 expression as the basis for leukemia classification.

[0067] 1. Cultivation of leukemia cells.

[0068] NB4, HL-60, THP-1, OCI-AML2 and OCI-AML3 cells were seeded in 6-well plates at appropriate cell numbers and cultured in an incubator at 37°C and 5% CO2. The cells were collected after 24 h for subsequent experiments.

[0069] 2. Use GPX4 expression level detection reagent to detect the expression level of GPX4.

[0070] 2-1 Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression level of GPX4 gene.

[0071] (1) Washing cells: Collect the cultured cells and centrifuge at 1500 rpm × 5 min; discard the supernatant, wash the cells with pre-cooled PBS, centrifuge at 5000 rpm × 4 min and discard the supernatant, repeat twice, discard the supernatant, and keep the cell pellet for later use.

[0072] (2) RNA extraction: RNA was extracted using the RNA-Quick Purification Kit. Add 250 µL Lysis Buffer to the obtained cell pellet and mix by pipetting; then add 250 µL anhydrous ethanol and mix by pipetting; add the liquid to the centrifuge column, centrifuge at 12000×g for 1 min, and discard the liquid in the collection tube; add 500 µL Wash buffer to the centrifuge column, centrifuge at 12000×g for 1 min, and discard the liquid in the collection tube; centrifuge the empty tube at 12000×g for 2 min to remove any residual Wash buffer as much as possible; take out the centrifuge column and place it in a clean 1.5 mL centrifuge tube without RNase, open the lid and dry for 6 min; add 20 µL Elution buffer to the center of the centrifuge column membrane and let it stand at room temperature for 2 min; centrifuge at 12000×g for 2 min, add the liquid in the 1.5 mL centrifuge tube to the center of the centrifuge column membrane again, let it stand for 2 min, centrifuge at 12000×g for 2 min again, discard the centrifuge column, and obtain the extracted RNA.

[0073] (3) Reverse transcription.

[0074] Prepare the reverse transcription system according to Table 1 and perform reverse transcription.

[0075] Table 1 Reverse transcription reaction system (20 μL system).

[0076]

[0077] Reaction conditions: 37°C × 15 min, 85°C × 5 s, 4°C cooling, cDNA stored at -40°C.

[0078] (4) qRT-PCR.

[0079] The real-time fluorescence quantitative PCR reaction system is shown in Table 2.

[0080] Table 2 qRT-PCR reaction system.

[0081]

[0082] Reaction conditions: First stage, 95°C pre-denaturation for 30 s; second stage, 95°C denaturation for 5 s, 58°C annealing for 30 s, 72°C full extension for 20 s, fluorescence collection, 39 cycles. Melting curve conditions: (65°C-95°C) fluorescence collection once every 0.5°C rise.

[0083] β-actin was used as the internal reference, and the relative quantitative value results were obtained using The primers of each target gene were synthesized by Shanghai Bioengineering Co., Ltd., and the sequences are shown in Table 3.

[0084] Table 3 Primer sequences for qRT-PCR.

[0085]

[0086] 2-2 Western blotting was used to detect the expression level of GPX4 protein.

[0087] (1) Washing cells: Collect the cultured cells and centrifuge at 1500 rpm × 5 min; discard the supernatant, wash the cells with pre-cooled PBS, centrifuge at 5000 rpm × 4 min and discard the supernatant, repeat twice, discard the supernatant, and keep the cell pellet for later use.

[0088] (2) Add protein lysis buffer (prepared with protease inhibitors and RIPA lysis buffer at a ratio of 1:100) approximately 4 times the volume of the precipitate and vortex thoroughly to mix; lyse the cells on ice for 30 min, vortex every 6 min, repeat 4 times, and centrifuge at 13,300 rpm for 30 min at 4°C; aspirate the supernatant solution into a pre-cooled new EP tube, detect the protein concentration by the BCA method, add approximately 1 / 4 of the protein sample volume of 5× loading buffer, and then boil and denature for 5 minutes, and finally store at −80°C.

[0089] (3) SDS-PAGE gel electrophoresis: Prepare gel according to the formula in Tables 4 and 5. First prepare the lower layer of 12% separation gel, add an appropriate amount of anhydrous ethanol to flatten it, and let it stand at room temperature for 30 minutes to solidify; pour out the anhydrous ethanol, and then prepare the upper layer of 5% concentration gel, insert the ruler comb, and let it stand at room temperature for 25 minutes to solidify. Fill the electrophoresis tank with electrophoresis buffer and set aside. When preparing for electrophoresis, remove the ruler comb and load samples as needed.

[0090] The proteins were separated by two-step electrophoresis.

[0091] Step 1: voltage 80 V, current 120 mA, 30 min;

[0092] Step 2: voltage 150 V, current 200 mA, 75 min.

[0093] Table 4 Separation gel recipe.

[0094]

[0095] Table 5. Stacking gel recipe.

[0096]

[0097] (4) Transfer: According to the molecular weight of the protein to be tested, cut a polyvinylidene fluoride (PVDF) membrane that matches it. The cut PVDF membrane is placed in methanol for 30 seconds for activation, washed with double distilled water for 2 minutes, and then soaked in pre-cooled wet transfer buffer. After the electrophoresis is completed, cut the gel in the area where the protein to be tested is located, and finally transfer the membrane in the order of sponge, filter paper, PVDF membrane, gel, filter paper and sponge from the positive electrode to the negative electrode. The membrane is transferred by constant current electrophoresis at 220 mA, and the transfer time is determined by the molecular weight of the protein.

[0098] (5) Blocking: After transfer, place the PVDF membrane in 5% protein blocking solution and block at room temperature for 2 h.

[0099] (6) Antibody incubation: Wash with TBST to remove the residual blocking solution on the membrane; use qualitative filter paper to absorb the residual liquid on the membrane, place the membrane on a wax plate, evenly cover the PVDF membrane with a certain proportion of pre-diluted primary antibody working solution, and incubate at 4°C overnight; after recovering the primary antibody working solution, place the PVDF membrane in TBST and wash on a shaker three times, each time for 10 min; finally, add a certain proportion of pre-diluted horseradish peroxidase-labeled secondary antibody to the PVDF membrane and incubate at room temperature for 75 min; after the incubation, wash three times with TBST, each time for 10 min.

[0100] (7) Color development and imaging: In a dark room, place the PVDF membrane on a chemiluminescent plate, then add the pre-configured chemiluminescent reagent to evenly cover the PVDF membrane, and display the image in the imaging system.

[0101] The results are as follows Figure 1 As shown in the figure, the expression of GPX4 in different cells is different. In general, GPX4 is highly expressed in NPM1 mutant leukemia cells OCI-AML3. Compared with the other four leukemia cells, the GPX4 gene expression level of NPM1 mutant leukemia cells OCI-AML3 is the highest, and its expression level is significantly different from these cells; the results of protein immunoblotting experiments show that compared with the other four leukemia cells, the GPX4 protein expression level of OCI-AML3 cells is also the highest.

[0102] Example 2

[0103] This example, based on Example 1, studies the regulatory effect of NPM1 mutation on GPX4.

[0104] First, a puromycin-resistant lentivirus was constructed to knock down NPM1 mutant protein, and the lentivirus was transfected into OCI-AML3 cells. During the process, puromycin was used to screen the constructed knockdown cells, and a stable OCI-AML3 cell line with NPM1 mutant protein knockdown was constructed. Western blotting was used to detect the expression level of GPX4 in OCI-AML3 cells after knocking down NPM1 mutant protein.

[0105] The results are as follows Figure 2 As shown, after knocking down the NPM1 mutant protein, the expression of GPX4 in OCI-AML3 cells decreased, suggesting that there may be a certain regulatory relationship between NPM1 and GPX4, and NPM1 mutant leukemia cells are cells with high expression of GPX4.

[0106] Example 3

[0107] This example studies the inhibitory effects of different drugs on the proliferation of different leukemia cell lines.

[0108] The specific experimental process was as follows: NB4, HL-60, THP-1, OCI-AML2 and OCI-AML3 cells were cultured at 1.0×10 4The number of cells was inoculated in a 96-well plate, and 5 replicate wells were set in each group. The above leukemia cells were treated with different drugs in a concentration gradient for 48 h. The different drugs included: sulfasalazine (SSZ), erastin, and RAS selective lethal molecule 3 (RSL3). Then 10 μL of CCK-8 reagent was added to each well, mixed and cultured for 3 h, the absorbance (OD) value was detected at a wavelength of 450 nm, and the cell survival rate was calculated.

[0109] The results are as follows Figures 3 to 5 As shown, the survival rates of the five leukemia cells decreased with the increase of drug concentrations under the treatment of SSZ, Erastin and RSL3, which indicated that SSZ, Erastin and RSL3 all have the ability to inhibit the proliferation of the leukemia cells.

[0110] Compared with the other four leukemia cells, the lowest half inhibitory concentration (IC50) values ​​of OCI-AML3 cells with NPM1 mutation after treatment with different drugs were: 229.8 μM for SSZ, 3.81 μM for Erastin, and 0.3843 μM for RSL3, indicating that OCI-AML3 cells are more sensitive to these drugs.

[0111] In general, the effect of inhibiting the proliferation of leukemia cells will be better only when the concentrations of these three drugs are relatively high, but this will lead to greater drug toxicity and side effects. Figure 3-Figure 5 According to the results, IC50 represents the drug concentration required for the drug to inhibit 50% of the proliferation of cultured cells. Since the IC50 of SSZ is 229.8 μM, which is significantly higher than that of Erastin at 3.81 μM, it indicates that SSZ is safer for cells, and SSZ will be used for the subsequent experiments.

[0112] Example 4

[0113] This example studies the inhibitory effects of different drugs on normal cell proliferation.

[0114] Specific experimental process: Normal bone marrow stromal cells HS-5 and leukemia cells were respectively added at 1.0×10 4 Each group of cells was inoculated in a 96-well plate, with 5 replicate wells, and treated with different drugs (SSZ and Erastin) in a concentration gradient for 48 h. Then 10 μL of CCK-8 reagent was added to each well, mixed and cultured for 3 h, and the absorbance (OD) value was detected at a wavelength of 450 nm, and the cell survival rate was calculated.

[0115] The results are as follows Figure 6 to Figure 7As shown, compared with leukemia cells, SSZ and Erastin had no effect on the proliferation of normal bone marrow stromal cells HS-5. Further comparison of the survival rate of HS-5 cells after treatment with different drugs showed that Erastin was more toxic to normal cells, so Erastin was not selected for subsequent experiments.

[0116] Example 5

[0117] Based on Example 3 and Example 4, this example detects the inhibitory effects of different drugs and drug compositions on the proliferation of OCI-AML3 cells.

[0118] Experimental steps: 1.0×10 4 OCI-AML3 cells were inoculated in 96-well plates, with 5 replicate wells in each group. First, OCI-AML3 cells were treated for 48 h with different drugs: RSL3 (0.1 µM), SSZ (100 µM), and small molecule anti-tumor drugs, and drug compositions of combined drug combinations (as shown in Table 6), respectively. The small molecule anti-tumor drugs were cytarabine (Ara-C, 0.5 µM), daunorubicin (DNR, 50 nM), and venetoclax (VEN, 2 µM); then, 10 μL of CCK-8 reagent was added to each well, mixed and cultured for 3 h; finally, the absorbance (OD) value was detected at a wavelength of 450 nm, and the cell survival rate was calculated.

[0119] It is noteworthy that the drug concentrations used in all pharmaceutical compositions were consistent with those of the corresponding single drugs.

[0120] Table 6 Pharmaceutical composition.

[0121]

[0122] The results are as follows Figures 8 to 10 As shown, overall, the inhibitory effect of the drug combination on the proliferation activity of OCI-AML3 cells is stronger than that of the single-drug treatment group, and the effect of low-dose single-drug inhibition of proliferation is poor. Furthermore, when RSL3 and SSZ are used in combination with chemotherapeutic drugs at the same time, the inhibitory effect on the proliferation activity of OCI-AML3 cells is stronger than that of the double-drug combination group of RSL3 or SSZ and chemotherapeutic drugs. Specifically, when RSL3 and SSZ are combined with Ara-C, the survival rate of OCI-AML3 cells is 61%; when RSL3 and SSZ are combined with DNR, the survival rate of OCI-AML3 cells is 46.9%; when RSL3 and SSZ are combined with VEN, the survival rate of OCI-AML3 cells is 55.2%. The above results show that low-dose RSL3 and SSZ combined with conventional chemotherapeutic drugs or new molecular targeted drugs can effectively kill OCI-AML3 cells and improve the therapeutic effect.

[0123] Furthermore, the effect of RSL3 and SSZ combined on the activity of NPM1 mutant leukemia cells was examined. OCI-AML3 cells were treated with different concentrations of RSL3 (0 µM, 0.1 µM, 0.2 µM) and SSZ (0 µM, 100 µM, 200 µM) separately or in combination for 48 h, and then CCK-8 was used to detect the cell activity of different treatment groups ( Fig.11 ), and the combined effect between RSL3 and SSZ was analyzed using the SynergyFinder website ( Fig.12 ).

[0124] Compared with the RSL3 and SSZ single-drug treatment groups, the same concentration of RSL3 combined with SSZ had a stronger inhibitory effect on the activity of OCI-AML3 cells, and there were significant differences. With the increase of the single-drug concentration of RSL3 / SSZ in the combined application, the inhibitory effect on the activity of OCI-AML3 cells became stronger and stronger. The results of SynergyFinder analysis also showed that the synergistic score of RSL3 and SSZ was 33.873, suggesting that RSL3 and SSZ played a synergistic effect in inhibiting the activity of OCI-AML3 cells.

[0125] It can be seen that compared with other dual-drug combinations, the combination of RSL3 and SSZ may have a more significant inhibitory effect on the proliferation of NPM1 mutant leukemia cells due to their synergistic effect. It further shows that the reason why RSL3 and SSZ combined with anti-tumor small molecule drugs have a better inhibitory effect on the proliferation of NPM1 mutant leukemia cells may be that the combination of RSL3 and SSZ has a synergistic effect, which further amplifies the killing effect of anti-tumor small molecule drugs on NPM1 mutant leukemia cells.

[0126] The combination of drugs in this embodiment includes three types, one is RAS selective lethal molecule 3 and sulfonamide antibiotics, one is sulfonamide antibiotics and anti-tumor small molecule drugs, and the other is RAS selective lethal molecule 3 and anti-tumor small molecule drugs. Specifically, the anti-tumor small molecule drug is selected from one or more of cytarabine, daunorubicin and venetoclax. In some preferred embodiments, the combination of drugs is RAS selective lethal molecule 3 and sulfonamide antibiotics.

[0127] In this embodiment, sulfasalazine is preferably used as the sulfonamide antibiotic. It is understandable that this embodiment can also be implemented by using sulfamethoxazole or sulfamethoxazole.

[0128] Furthermore, as a preferred method, the combination drug combination is preferably a three-drug combination, and the RAS selective lethal molecule 3 and sulfonamide antibiotics combined with anti-tumor small molecule drugs have a better inhibitory effect on the proliferation of NPM1 mutant leukemia cells.

[0129] Example 6

[0130] This example studies the inhibitory effect of RSL3 on the expression level of GPX4 in OCI-AML3 cells.

[0131] This example mainly uses a GPX4 expression level detection reagent to evaluate the expression level of GPX4 in NPM1 mutant leukemia cells OCI-AML3 after RSL3 treatment. The specific experimental method is similar to that of Example 1, except that OCI-AML3 cells need to be treated with 0.4 μM RSL3 for 48 h, and a control group without RSL3 treatment is set up.

[0132] The results are as follows Fig.13 As shown, compared with untreated cells, the GPX4 gene expression level of OCI-AML3 cells treated with 0.4 µM RSL3 was significantly reduced by 2.25 times (A), and the protein level was also reduced by 1.15 times (B), proving that RSL3 can inhibit the gene expression and protein levels of GPX4 in OCI-AML3 cells, suggesting that RSL3 can inhibit the proliferation of NPM1 mutant leukemia cells that highly express GPX4 by inhibiting the level of GPX4.

[0133] Example 7

[0134] This example studies the relationship between the GPX4 expression level in leukemia cells and drug sensitivity.

[0135] The experimental process is: gray value quantification of the GPX4 protein band of the leukemia cell line obtained in Example 1, and then correlation analysis of the protein quantification result and the IC50 value of the leukemia cell line SSZ drug obtained in Example 3.

[0136] The results are as follows Fig.14 As shown in the figure, the higher the GPX4 protein level in leukemia cells, the higher the sensitivity to SSZ and the smaller the IC50 value of the drug used, indicating that the GPX4 expression level in leukemia cells is negatively correlated with the sensitivity to SSZ. In other words, using a low dose of SSZ on leukemia cells with high GPX4 expression can achieve a better therapeutic effect with fewer side effects.

[0137] Example 8

[0138] This example provides validation of ferroptosis inhibitors to assess the level of ferroptosis induced by SSZ in OCI-AML3 cells.

[0139] The specific process is as follows: OCI-AML3 cells were plated at 1.0×10 4The number of cells was inoculated in a 96-well plate, and 5 replicate wells were set up in each group. OCI-AML3 cells were treated with 200 μM SSZ and ferrostatin-1 (Fer-1), apoptosis inhibitor Z-VAD-FMK (Z-VAD), necrosis inhibitor necrostatin-1 (Necro-1) and autophagy inhibitor chloroquine (CQ) for 48 h, respectively; then 10 μL CCK-8 reagent was added to each well, mixed and cultured for 3 h; the absorbance (OD) value was detected at a wavelength of 450 nm.

[0140] In this embodiment, ferrostatin-1 is preferably used as the ferroptosis inhibitor. It is understood that this embodiment can also be implemented by using liproxstatin-1, troglitazone or zileuton.

[0141] This example uses CCK-8 experiment to observe the effect of different death inhibitors on SSZ inhibition of OCI-AML3 cell activity. Fig.15 As shown in the figure, only Fer-1 partially reversed the inhibitory effect of SSZ on the proliferation of OCI-AML3 cells, suggesting that SSZ inhibited the proliferation of NPM1 mutant leukemia cells by inducing ferroptosis. However, the degree of ferroptosis induced by low-dose SSZ was small and insufficient to induce the death of a large number of NPM1 mutant leukemia cells, resulting in limited therapeutic effects. Therefore, RSL3 was used in combination to achieve better leukemia cell killing.

[0142] Example 9

[0143] This embodiment provides an example of the use of the above-mentioned pharmaceutical composition in the preparation of a reagent for evaluating the ferroptosis effect of NPM1 mutant leukemia.

[0144] The pharmaceutical composition comprises RAS selective lethal molecule 3 and sulfonamide antibiotics.

[0145] Wherein, the sulfonamide antibiotic is selected from at least one of sulfasalazine, sulfadiazine, sulfamethoxazole and sulfadimethoxine.

[0146] The NPM1 mutant leukemia ferroptosis effect evaluation reagent includes a ferroptosis inhibitor and a GPX4 expression level detection reagent.

[0147] Wherein, the ferroptosis inhibitor is used to evaluate the degree of ferroptosis induced by the sulfonamide antibiotics.

[0148] The GPX4 expression level detection reagent may include a real-time fluorescence quantitative PCR reagent and / or a protein immunoblotting test reagent, which is used to evaluate the expression level of GPX4 in NPM1 mutant leukemia cells inhibited by RAS selective lethal molecule 3.

[0149] Furthermore, the NPM1 mutant leukemia ferroptosis effect evaluation reagent also includes experimental cells, and the experimental cells are NPM1 mutant leukemia cells OCI-AML3 with high expression of GPX4.

[0150] The pharmaceutical composition is configured in unit dose in the NPM1 mutant leukemia ferroptosis effect evaluation reagent.

[0151] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0152] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A combination drug for inhibiting the proliferation of NPM1 mutant leukemia cells, characterized in that: The combined drug combination consists of RAS selective lethal molecule 3 and sulfonamide antibiotics or consists of RAS selective lethal molecule 3, sulfonamide antibiotics and anti-tumor small molecule chemical drugs; the sulfonamide antibiotic is sulfasalazine.

2. The combination drug according to claim 1, characterized in that: The anti-tumor small molecule drug is selected from at least one of cytarabine, daunorubicin and venetoclax.

3. The combined drug composition according to claim 1, characterized in that: The NPM1 mutant leukemia cells are cells with high expression of GPX4.

4. The combined drug composition according to claim 1, characterized in that: The effective starting concentration of the RAS selective lethal molecule 3 in the combined drug combination is not less than 0.1 μM; the effective starting concentration of the sulfonamide antibiotic is not less than 100 μM.

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